The covalent attachment of polyethylene glycol (PEG) moieties to proteins or polypeptides (“PEGylation”) is a well-known technique for improving the properties of such proteins or polypeptides, in particular pharmaceutical proteins, e.g. in order to improve circulation half-life and/or to shield potential epitopes and thus reduce the potential for an undesired immunogenic response. Numerous technologies based on activated PEG are available to provide attachment of the PEG moiety to one or more groups on the protein. For example, mPEG-succinimidyl propionate (mPEG-SPA, available from Nektar Therapeutics) is generally regarded as being selective for attachment to the N-terminus and epsilon-amino groups of lysine residues via an amide bond. However, in practice mPEG-SPA does not always attach exclusively to these groups, but may also attach to the hydroxyl group of a serine, tyrosine or threonine residue via an ester bond. As a result, PEGylated proteins prepared using this technology may not have a sufficient degree of uniformity and may contain a number of different PEG isomers other than those that were intended. This is undesired for various reasons, including the fact that it makes characterization of such proteins more complicated. Further, PEG moieties bound to groups other than those intended may be relatively unstable. For example, PEG moieties bound to a hydroxyl group via an ester bond in the case of an amine-specific PEG such as mPEG-SPA tend to be relatively unstable compared to those moieties that are bound via an amide bond to the N-terminal or a lysine residue. Depending on the formulation and storage conditions, this may lead to an undesired loss of these labile PEG groups and thus potentially to a change in the properties of the PEGylated protein over time. Further, there may be potential regulatory or safety issues for a PEGylated protein pharmaceutical in which there is a risk that one or more of the PEG moieties may detach from the protein in the body after it is administered to a patient.
The present invention addresses these problems by providing a method by which such labile PEG groups may be easily removed, thereby resulting in a more uniform and stable PEGylated protein product.